PURIFICATION AND CHARACTERIZATION OF LACCASES FROM THE WHITE-ROT BASIDIOMYCETE DICHOMITUS SQUALENS

Two chromatographic forms of laccase c1 and c2 were purified approxima tely 225-fold from the extracellular culture fluid of ligninolytic cul tures of Dichomitus squalens, using DEAE-Sepharose and Mono-Q fast pro tein liquid chromatography. Each homogeneous laccase (cl and c2) has a molecular mass of approximately 66 kDa as determined by SDS-PAGE. Bot h forms are glycoproteins, and each contains four copper atoms per mol ecule of:protein. The first 20 amino acids of the N-terminal sequences of these two laccases are identical and are similar to those of lacca ses from other lignin-degrading fungi, The electronic absorption spect ra of these laccases exhibit bands at 610 and 330 nm, indicative of ty pe I and type III copper. The EPR spectrum Of laccase cl exhibits band s indicative of type I and type II copper. Each laccase oxidizes a var iety of phenolic substrates, has a pH optimum of 3.0 for the oxidation of 2,6-dimethoxyphenol, and is inhibited strongly by fluoride and azi de. (C) 1998 Academic Press.